Transcriptional reprogramming of infiltrating neutrophils drives lung disease in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. Nevertheless, targeted studies within this vulnerable population are scant. Here, we applied multi-omics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, led to a persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of a transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to ARDS in a well-defined patient population disproportionally affected by severe COVID-19.

REALITI-A Study: Real-World Oral Corticosteroid-Sparing Effect of Mepolizumab in Severe Asthma.

BACKGROUND: Patients with severe asthma may require maintenance oral corticosteroids (mOCS) for disease control as well as systemic corticosteroid (SCS) bursts for clinically significant exacerbations. However, mOCS and SCS use are associated with adverse effects, which increases patient disease burden. OBJECTIVE: To assess the real-world corticosteroid-sparing effect of mepolizumab in patients with severe asthma. METHODS: REALITI-A was a 24-month international, prospective, observational cohort study involving 84 centers across Europe, Canada, and the United States, with a 1-year pre-post mepolizumab treatment preplanned interim analysis. A total of 822 adults with a clinical diagnosis of asthma and a physician decision to initiate mepolizumab treatment (100 mg subcutaneously) were included. End points included daily mOCS dose at baseline (penultimate 28 days of pretreatment) and 1 year after treatment; percent reduction from baseline in mOCS dose; patients discontinuing mOCS 1 year after treatment; and the rate of clinically significant exacerbations (those requiring OCS for 3 days or more [or parenteral administration], emergency room visit, and/or hospital admission) before and after treatment. RESULTS: A total of 319 patients received mOCS at baseline (median [interquartile range]: 10.0 [5.0-15.0] mg/d). At 1 year after treatment, median mOCS dose was reduced by 75% (2.5 [0.0-5.0] mg/d); 64% of patients had a reduction in mOCS dose of 50% or greater compared with baseline and 43% discontinued mOCS. Clinically significant exacerbations decreased between pretreatment and posttreatment (rate ratio [95% confidence interval] 0.29 [0.26-0.32]; P < .001). CONCLUSION: This 1-year analysis demonstrates that real-world mepolizumab treatment is clinically effective in patients with severe asthma, providing disease control while reducing the need for mOCS and SCS bursts.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, as a dominant feature of severe and critical COVID-19 (refs. 14-18). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Delayed Kinetics of IgG, but Not IgA, Antispike Antibodies in Transplant Recipients following SARS-CoV-2 Infection.

BACKGROUND: Kidney transplant recipients are at increased risk of severe outcomes during COVID-19. Antibodies against the virus are thought to offer protection, but a thorough characterization of anti-SARS-CoV-2 immune globulin isotypes in kidney transplant recipients following SARS-CoV-2 infection has not been reported. METHODS: We performed a cross-sectional study of 49 kidney transplant recipients and 42 immunocompetent controls at early (≤14 days) or late (>14 days) time points after documented SARS-CoV-2 infection. Using a validated semiquantitative Luminex-based multiplex assay, we determined the abundances of IgM, IgG, IgG1-4, and IgA antibodies against five distinct viral epitopes. RESULTS: Kidney transplant recipients showed lower levels of total IgG antitrimeric spike (S), S1, S2, and receptor binding domain (RBD) but not nucleocapsid (NC) at early versus late time points after SARS-CoV-2 infection. Early levels of IgG antispike protein epitopes were also lower than in immunocompetent controls. Anti-SARS-CoV-2 antibodies were predominantly IgG1 and IgG3, with modest class switching to IgG2 or IgG4 in either cohort. Later levels of IgG antispike, S1, S2, RBD, and NC did not significantly differ between cohorts. There was no significant difference in the kinetics of either IgM or IgA antispike, S1, RBD, or S2 on the basis of timing after diagnosis or transplant status. CONCLUSIONS: Kidney transplant recipients mount early anti-SARS-CoV-2 IgA and IgM responses, whereas IgG responses are delayed compared with immunocompetent individuals. These findings might explain the poor outcomes in transplant recipients with COVID-19.

One-Stop Serum Assay Identifies COVID-19 Disease Severity and Vaccination Responses.

SARS-CoV-2 has caused over 100,000,000 cases and almost 2,500,000 deaths globally. Comprehensive assessment of the multifaceted antiviral Ab response is critical for diagnosis, differentiation of severity, and characterization of long-term immunity, especially as COVID-19 vaccines become available. Severe disease is associated with early, massive plasmablast responses. We developed a multiplex immunoassay from serum/plasma of acutely infected and convalescent COVID-19 patients and prepandemic and postpandemic healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 nucleocapsid (N), spike domain 1 (S1), S1-receptor binding domain (RBD) and S1-N-terminal domain. For diagnosis, the combined [IgA + IgG + IgM] or IgG levels measured for N, S1, and S1-RBD yielded area under the curve values ≥0.90. Virus-specific Ig levels were higher in patients with severe/critical compared with mild/moderate infections. A strong prozone effect was observed in sera from severe/critical patients-a possible source of underestimated Ab concentrations in previous studies. Mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared with severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 mo after symptom onset. Measurement of the Ab responses in sera from 18 COVID-19-vaccinated patients revealed specific responses for the S1-RBD Ag and none against the N protein. This highly sensitive, SARS-CoV-2-specific, multiplex immunoassay measures the magnitude, complexity, and kinetics of the Ab response and can distinguish serum Ab responses from natural SARS-CoV-2 infections (mild or severe) and mRNA COVID-19 vaccines.

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

Antibody Responses to SARS-CoV-2: Let's Stick to Known Knowns.

The scale of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has thrust immunology into the public spotlight in unprecedented ways. In this article, which is part opinion piece and part review, we argue that the normal cadence by which we discuss science with our colleagues failed to properly convey likelihoods of the immune response to SARS-CoV-2 to the public and the media. As a result, biologically implausible outcomes were given equal weight as the principles set by decades of viral immunology. Unsurprisingly, questionable results and alarmist news media articles have filled the void. We suggest an emphasis on setting expectations based on prior findings while avoiding the overused approach of assuming nothing. After reviewing Ab-mediated immunity after coronavirus and other acute viral infections, we posit that, with few exceptions, the development of protective humoral immunity of more than a year is the norm. Immunity to SARS-CoV-2 is likely to follow the same pattern.

Antibody Profiles According to Mild or Severe SARS-CoV-2 Infection, Atlanta, Georgia, USA, 2020.

Among patients with coronavirus disease (COVID-19), IgM levels increased early after symptom onset for those with mild and severe disease, but IgG levels increased early only in those with severe disease. A similar pattern was observed in a separate serosurveillance cohort. Mild COVID-19 should be investigated separately from severe COVID-19.